This invention relates to a process for producing interferon and more particularly to an improved method for the production of interferon by treating animal cells proliferated on a positively charged microcarrier with an interferon inducer.
Interferon is a protein produced by animal cells by the stimulus of an inducer such as a variety of viruses or double-stranded RNA which is active in inhibiting intracellular proliferation of viruses. Whereas interferon is non-specific for virus species, it is specific for animal species. In other words, an interferon produced with cells of an animal species is not active on another species. In recent years, possible use of interferon as a medicinal has called attention since therapeutic effects of interferon on certain virus diseases and tumors were discovered. In order to achieve application of interferon as a medicinal it is necessary to implement in vitro production, isolation and purification of interferon using a member of human cells. Leucocyte separated from blood was often employed as the cell for producing interferon, but more recently, foetal or neonatal diploid cells have been used instead. Supply of leucocyte necessarily depends upon a group of a large number of non-specified persons, and the interferon products thus prepared will highly possibly be contaminated with various lymphokines. It is therefore difficult to assure safety of the interferon products obtained from leucocyte. On the other hand, as it is easy in the case of the diploid cells to cultivate on a large scale the cells originated from an individual, it is considered that assurance of safety is easilier for such interferon products then for those from leucocyte.
It is known to use a Roux bottle or a roller bottle for the cultivation of anchorage dependent cells such as diploid cells. However, cultivation of a large amount of anchorage dependent cells by such method is considered rather difficult. This is because cultivation on a large scale by this method in which the cells proliferate in monolayer on the bottom of a Roux bottle or on the side of a roller bottle needs a large number of Roux bottles or roller bottles to be handled.
A. L. van Wezel found that animal cells can attach to and proliferate on DEAE Sephadex A50, and named this culture method as microcarrier culture. But DEAE Sephadex A50 has some problems as microcarrier because it has toxic effect on cell growth. D. W. Levine et al improved cell growth by addition of negatively charged polymer such as carboxymethylcellulose to DEAE Sephadex A50 (see U.S. Pat. No. 4,036,693). Further, Levine et al found diethylaminoethylated bridged dextran with lower charge density than DEAE Sephadex A50 is good microcarrier for cultivating anchorage dependent cell and developed a culture method which is suitable for the large-scale cultivation of anchorage dependent cells (see U.S. Pat. No. 4,189,534).
The method consists of inoculating seed cells in a medium composed of a suspended positively charged microcarrier (called microcarrier for short hereinbelow) and carrying out a suspension culture. The inoculated cells attached to surfaces of the microcarrier on which the cells proliferated. The microcarrier culture method readily increases the surface area on which the cells are attached and is easy to handle. The method therefore can be regarded as a culture method quite suitable for the large-scale cultivation of anchorage dependent cells.
In addition to the above-mentioned technique by which interferon is produced in culture cells, there are known a method called superinduction method in which production of interferon is further enhanced by stimulating the cells with an interferon inducer such as double-stranded RNA followed by treatment of the stimulated cells with an antimetabolite such as cycloheximide or actinomycin D (see U.S. Pat. No. 3,773,924) and a method called UV method in which the production of interferon is further enhanced by irradiating the cells with UV light shortly before and/or after the stimulation with an interferon inducer.
It is also known as a priming effect of interferon that treatment of the cells with an interferon at a low concentration stabilizes production of the interferon.
In consideration of the above-described culture and interferon production methods for producing a large amount of interferon from cultured cells such as diploid cells, we carried out a production of interferon by the superinduction method after priming interferon treatment of the cells cultivated by the microcarrier culture method. Such method often produced a low-potency interferon and was not stable in producing a high-potency interferon.